Fig. 3.

OspD2 regulates the levels of proteins translocated into the cytosol of infected cells. (A and B) Secretion assays of Shigella as described in Fig. 1. Membranes were immunoblotted with the designated antibodies. The upper fragment of SDS/PAGE gels of supernatant fractions were silver-stained to visualize SepA, the loading control. (C) Immunoblots of FLAG-tagged effectors expressed under the control of their endogenous VirB-regulated promoters present in Shigella grown under conditions that induce T3SS expression, but not activation. (D and E) HeLa cells infected at an MOI of 30 with Shigella strains expressing Afa-1 at designated time points were treated with RIPA buffer to lyse mammalian, but not bacterial, cell membranes. The soluble and insoluble (bacterial-containing) fractions were separated and immunoblotted with designated antibodies. GroEL served as a fractionation and loading control for the insoluble fraction, and β-actin served as a loading control for the soluble fraction. Each immunoblot is representative of at least three experimental repeats.